Gs is the heterotrimeric G protein responsible for stimulating adenylyl cyclase. Activation of Gs occurs when an agonist-receptor complex promotes the exchange of GDP for GTP in the guanine nucleotide binding site of the a subunit (Gsa). GTP analogs such as Gpp[NH]p also activate Gs. Activation of Gs leads to conformational changes, and can cause dissociation of Gsa from the bg-subunit complex. It has been proposed that subunit dissociation necessarily accompanies Gs activation. We developed a method for doing rate zonal sedimentation in approximately one hour and used it to investigate the effects of activation on Gs. Unactivated Gs and the free Gsa had sedimentation coefficients of approximately 3.8 S and 2.3 S, respectively, whereas that of Gpp[NH]p-activated Gs was approximately 3.3 S. Equilibrium sedimentation experiments indicated that the molecular weight of Gpp[NH]p-activated Gs was equivalent to that of a Gs heterotrimer. Thus, the change in sedimentation coefficient following activation by Gpp[NH]p was probably due to large conformational changes rather than to subunit dissociation. These data corroborate other data from this laboratory supporting our model that subunit dissociation does not necessarily accompany Gs activation. Inactivating mutations in Gsa are the molecular basis for Albright hereditary osteodystrophy (AHO), a human disease typified by resistance to hormonal stimulation of adenylyl cyclase. Three separate point mutations (525 OR, R258W, and E259V) and one codon deletion (N98) in the gene for Gsa have been identified in patients with AHO. Each mutant was cloned and studied after translation in vitro; and with the exception of N98, was stably expressed in 549 cyc- cells which lack Gsa. Both S250R and E259V were thermolabile at physiological temperatures, and located predominantly in the cytosol rather than in the membranes of transfected 549 cyc- cells. Membranes from 549 cyc- cells transformed with R258W displayed normal activity when activated with GTPgS, but responded weakly to both fluoraluminate and isoproterenol. Activation of Gs protects the a subunit from proteolytic degradation, and protease protection assays suggest that all of the point mutations caused a reduction in the affinity of Gsa for guanine nucleotides. Gsa is unstable unless a guanine nucleotide is bound to its nucleotide binding site, and the instability of the a subunits that harbor the point mutations is probably due to their reduced affinity for guanine nucleotides. N98 was inactive in all assays performed thus far. The three dimensional structure of several G protein a subunits have been determined by X-ray crystallography. Computer modeling based on these structures can be used to predict changes in the secondary structure that result from changes in the primary structure. Currently, computer modeling is being used in a effort to develop a hypothesis to explain the lack of biological activity in the N98 mutant.